Plants are dependent upon light as a signal for regulating both developmental and metabolic processes, and phytochrome is held to be the principal plant photoreceptor that regulates these responses. The long- term objective of our laboratory is to identify the components of phytochrome signaling pathways and to understand how these pathways are regulated. By developing a microinjection-based experimental system, we have demonstrated the existence of three signal transduction pathways that are downstream of one of the phytochromes (PHYA), and which regulate the expression of genes encoding chloroplast components and anthocyanin biosynthetic enzymes. One pathway is dependent upon calcium, the second upon cGMP, and the third upon both of these effectors. We have established CAB, CHS, and FNR, as specific reporter genes for the activity of each of the three PHYA signaling pathways respectively. We now propose to investigate these pathways further using four approaches: A) To identify individual cis-elements within the CAB, CHS and FNR promoters that can respond to calcium and/or cGMP. This will be carried out by microinjection experiments using various promoter deletions and synthetic promoters containing one or more previously identified cis-elements. Our attention will be focused on binding sites for the transcription factor GT-1 in the CAB promoter, and on G-boxes and Myb-factor binding sites within the CHS promoter. B) In addition to the PHYA signaling intermediates already elucidated, we hope to use our microinjection techniques to characterize others. In particular, we will examine the possible participation of calcium/calmodulin-dependent protein kinase II and guanylyl cyclase. C) Using a mutant isolation screen in Arabidopsis based upon the aberrant expression of a photoregulated reporter gene (CAB- LUC) in response to light, we have isolated a number of mutants which may be altered in PHYA signal transduction. The photobiology and PHYA dependency of mutant phenotypes will be characterized and those clearly affected in PHYA signal transduction will be analyzed to determine the sites of lesions within the signaling pathways. D) Based upon recent experiments e now have some understanding of how individual PHYA signaling pathways are regulated, and how cross-talk mechanisms between each pathway operate. We have devised new screening strategies to isolate mutants in such regulatory mechanisms, based upon the aberrant expression of the CAB- LUC reporter gene or a CHS-LUC reporter gene which has recently been inserted into the Arabidopsis genome in our laboratory. Using these approaches we hope to expand our knowledge not only of the processes by which plants perceive light via phytochrome, but also the regulatory processes which operate to coordinate the expression of genes regulated by phytochrome.